PROJECT SUMMARY/ABSTRACT Despite recent advances in the targeting of immune checkpoints across malignancies, many patients fail to respond to treatment, suggesting alternative mechanisms of immunosuppression. Metabolic dysfunction within tumor-infiltrating T-cells has emerged as a potential mechanism by which long-term anti-tumor immunity is impaired. The mechanism by which altered metabolism suppresses intratumoral T-cell function, however, remains to be fully characterized. Cellular metabolites not only supply the bioenergetics needs of proliferating immune cells, but also regulate gene expression by serving as the substrates for chromatin modifications. Preliminary data presented in this proposal utilize a combination of in vitro and in vivo systems to explore the metabolic liabilities of T-cells during chronic exposure to tumor antigens. Under these conditions, T-cells experience high levels of oxidative stress. This compromises the ability of T-cells to oxidize glucose and glutamine within the TCA cycle, leading to bioenergetic compromise that impairs nucleotide biosynthesis and alters the availability of substrates for DNA and histone demethylation reactions. Enhancing redox homeostasis has beneficial effects on anti-tumor T-cell immunity in vitro as well as in vivo. Thus, the efficacy of T-cells within the tumor microenvironment may be primarily limited by metabolic alterations that generate redox stress. This hypothesis will be rigorously tested by (1) using a combination of in vitro and in vivo models of T-cell exhaustion in both mice and primary patient tumors to define the impact of chronic antigen-driven metabolic alterations on T-cell proliferation, chromatin modifications, and effector function, (2) determining how cysteine limitation exacerbates T-cell dysfunction within the tumor microenvironment, and (3) determining whether enhancing redox homeostasis, either by increasing intracellular cysteine availability or limiting the generation of oxidative stress can reverse metabolic T-cell dysfunction and enhance anti-tumor immunity. The proposed investigations will expand the armamentarium of strategies to enhance immune responses in cancer, particularly for the patients who are unresponsive to anti-PD-1 therapy. The applicant, Dr. Santosha Vardhana, an Assistant Attending with the Lymphoma Service at Memorial Sloan Kettering Cancer Center, has outlined a 5-year career plan that builds on his scientific background in immunology and cellular metabolism as well as his clinical training in medical oncology and immunotherapy. Dr. Vardhana will conduct the proposed research under the mentorship of Dr. Craig Thompson, an internationally recognized expert in immunology and metabolism with a strong track record of training successful physician scientists, with co-mentorship by Dr. Jedd Wolchok, a highly recognized expert in cancer immunotherapy with significant experience interrogating T- cell function in both mouse models and primary patient samples. MSKCC provides the ideal institutional environment for Dr. Vardhana to embark on the proposed research program and transition to a position as an independent academic investigator with his own laboratory and R01 funding.